Pluripotent stem cells hold enormous promise for biomedicine. Indeed the recent development of induced pluripotent stem cell (iPSC) procedures allows the generation of patient-specific pluripotent cells from any individual. Induced pluripoten stem cells closely resemble their embryo-derived counterparts, embryonic stem cells (ESC). Currently, however, the exact degree of identity between ESC and iPSC is unclear. Human (h) iPSC are genetically identical to the person of origin and thus, carry the exact complement of genetic variables that cause or predispose the patient to disease. This enables the development of exciting and unprecedented avenues to model disease etiology, better diagnostic and pharmaceutical reagents and in time, defined mature cell populations suitable for transplantation-based therapies. In order to realize the potential of pluripotent cells a detailed, quantitative understanding of their regulatory mechanisms is critical. By their nature, complex biological systems cannot be understood using reductionist approaches. Rather, systems biology paradigms that merge global experimental technologies with computational approaches are required to reveal how a cell processes biological information to affect a change in fate. Together with our computational colleagues we have developed such a systems approach and applied it to study the regulation of mouse (m) ESC, the founding and best-characterized member of the pluripotency pantheon. In the current proposal we will extend our studies to define how biological information is processed by mESC over time after a defined perturbation. We will focus on transcriptional regulators such as Nanog, Esrrb and Tbx3, and others that are necessary for pluripotency and measure their functions at the epigenetic, transcriptional, mRNA, microRNA and proteomic levels. The interactions among these factors in regulatory modules will also be explored. These studies will provide an unprecedented dynamic view of ESC regulation. In a sense, how individual pluripotent cells traverse the Waddington Landscape will be measured and visualized. Other genetic loss-of-function studies will be pursued to identify the complete panel of protein-coding gene-products that together function to maintain the pluripotent state and its transitions. We will also apply novel analytical tools to identify molecules that alter ESC properties in subtle and previously undefinable ways. Selected candidate molecules have already emerged and will be studies in detail. Finally, the existence of multiple alternative pluripotency regulatory network configuration will be explored and the roles of biological noise as well as stochasticity in ESC regulation will be addressed. PUBLIC HEALTH RELEVANCE: This project will provide a quantitative and comprehensive view of how pluripotent stem cells process molecular information during fate decisions. Such a view is necessary for the realization of the enormous medical promises of pluripotent stem cells, and will serve as a firm foundation for the development of disease models, novel diagnostic tool as well as pharmaceutical compounds. Ultimately, these studies will also impact on the derivation of defined cell populations for regenerative medicine applications.